This competitive renewal application explores molecular mechanisms regulating vascular smooth muscle cell (VSMC) relaxation by the nitric oxide/cGMP/cGMP-dependent protein kinase (PKG) pathway. VSMC contractile state is dynamically regulated by phosphorylation of the regulatory myosin light (MLC) chains controlling actinomyosin contraction. Increases in [Ca2+]in activate the Ca 2+/calmodulin-dependent MLC kinase (MLCK), which phosphorylates MLC, causing VSMC contraction. Conversely, increases in myosin phosphatase (PP1M) activity, such as occurs in response to the endogenous vasodilator nitric oxide (NO), dephosphorylate MLC, causing relaxation. Over the past decade, we have focused on understanding molecular mechanisms regulating vascular relaxation by identifying new PKGIalpha targets and testing their functional role. PKGI induces VSMC relaxation by multiple mechanisms involving phosphorylation of substrates that directly regulate (i) actinomyosin contractile stress fiber relaxation and (ii) inhibition of GPCR- mediated [Ca+2]in mobilization. We have characterized several PKGIa-target protein interactions important in VSMC relaxation, including interactions with (a) PP1M, (b) the regulator of G protein signaling 2 (RGS2), and (c) the formin homology domain-containing protein type 1, or FHOD1, a multi-functional protein that stimulates VSMC stress fiber formation. All 3 of these pathways are mediated by PKGIalpha binding/activation of these targets via its N-terminal leucine zipper (LZ) interaction domain, supporting the hypothesis that PKGIalpha has a central role in the regulation of normal VSMC tone and biology via PKGIalpha-target protein interactions mediated by the PKGIalpha LZ domain. To explore this hypothesis, we now have created mice expressing a LZ mutant (LZM) PKGIalpha defective for protein-protein interactions, but otherwise identical to wild-type PKGIa. LZM 'knock-in'mice have an exciting phenotype supporting the above hypothesis: LZM VSMC have excessive actinomyosin stress fibers;LZM mouse blood vessels relax abnormally;and intact PKGIalpha LZM mice are hypertensive, providing exciting tools with which to explore the central hypothesis of this application. We propose to investigate: the molecular mechanisms by which PKGIa inhibits VSMC actinomyosin stress fiber formation and contraction, with a focus on the FHOD1-PKG LZ domain interaction (SA1);PKGIa inhibition of GPCR-mediated [Ca+2]in mobilization, with a focus on LZ-mediated interactions regulating the (i) RGS2-GPCR-IP3 pathway and (ii) thromboxane receptor (SA2);and the functional role of the PKGIalpha LZ targeting domain in vascular regulation in intact blood vessels and animals, using WT and LZM mice (SA3). These studies are expected to increase our understanding of vascular tone regulation, with the potential to advance the diagnosis and therapy of human cardiovascular diseases.